U.S. patent application number 10/389484 was filed with the patent office on 2004-01-08 for method for generating primate trophoblasts.
Invention is credited to Thomson, James A., Xu, Ren-He.
Application Number | 20040005701 10/389484 |
Document ID | / |
Family ID | 28042013 |
Filed Date | 2004-01-08 |
United States Patent
Application |
20040005701 |
Kind Code |
A1 |
Xu, Ren-He ; et al. |
January 8, 2004 |
Method for generating primate trophoblasts
Abstract
The first method to cause a culture of human and other primate
stem cells to directly and uniformly differentiate into a committed
cell lineage is disclosed. Treatment of primate stem cells with a
single protein trophoblast induction factor causes the cells to
transform into human trophoblast cells, the precursor cells of the
placenta. Several protein factors including bone morphogenic
protein 4 (BMP4), BMP2, BMP7, and growth and differentiation factor
5 can serve as trophoblast-inducting factors.
Inventors: |
Xu, Ren-He; (Madison,
WI) ; Thomson, James A.; (Madison, WI) |
Correspondence
Address: |
Nicholas J. Seay
Quarles & Brady LLP
P O Box 2113
Madison
WI
53701-2113
US
|
Family ID: |
28042013 |
Appl. No.: |
10/389484 |
Filed: |
March 14, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60365136 |
Mar 15, 2002 |
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Current U.S.
Class: |
435/363 ;
435/366 |
Current CPC
Class: |
C12N 5/0605 20130101;
C12N 2506/02 20130101; C12N 2501/155 20130101; C12N 2501/19
20130101 |
Class at
Publication: |
435/363 ;
435/366 |
International
Class: |
C12N 005/06; C12N
005/08 |
Claims
I/We claim:
1. A method to induce primate stem cells to differentiate into
human trophoblast cells comprising the step of culturing the
primate stem cells in the presence of a protein
trophoblast-inducting factor.
2. The method of claim 1 wherein the protein trophoblast-inducing
factor is selected from the group consisting of bone morphogenic
protein 4 (BMP4), bone morphogenic protein 2(BMP2), bone
morphogenic protein 7(BMP7) and growth and differentiation factor 5
(GDF5).
3. The method of claim 2 wherein the protein trophoblast-inducing
factor is applied to the stem cells at a concentration of between 1
and 100 nanogram per milliliter of culture medium.
4. The method of claim 1 wherein the stem cells are human embryonic
stem cells.
5. The method of claim 1 wherein the stem cells are non-human
primate embryonic stem cells.
6. A culture of primate trophoblast cells in in vitro culture, the
trophoblast cells derived from primate stem cells exposed to a
trophoblast inducing factor.
7. The culture of claim 6 wherein the trophoblast-inducing factor
is selected from the group consisting of bone morphogenic protein 4
(BMP4), bone morphogenic protein 2(BMP2), bone morphogenic protein
7(BMP7) and growth and differentiation factor 5 (GDF5).
8. The culture of claim 6 wherein the trophoblast cells are human
cells.
9. The culture of claim 6 wherein the trophoblast cells are
non-human primate cells.
10. A method of testing agents for their effect on placental cells
comprising creating trophoblast cells by culturing primate stem
cells in the presence of a trophoblast inducing factor; exposing
the trophoblast cells to the agent; and observing what effect the
agent has on the trophoblast cells.
11. The method of claim 10 wherein the trophoblast-inducing factor
is selected from the group consisting of bone morphogenic protein 4
(BMP4), bone morphogenic protein 2(BMP2), bone morphogenic protein
7(BMP7) and growth and differentiation factor 5 (GDF5).
12. The method of claim 10 wherein the trophoblast-inducing factor
is applied to the stem cells at a concentration of between 1 and
100 nanogram per milliliter of culture medium.
13. The method of claim 10 wherein the stem cells are human
embryonic stem cells.
14. The method of claim 10 wherein the stem cells are non-human
primate embryonic stem cells
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/365,136 filed Mar. 15, 2002.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not applicable.
BACKGROUND OF THE INVENTION
[0003] Modern cell biology includes a variety of techniques to
manipulate various cells of living organisms in vitro. Of
particular interest is a category of cell known as a stem cell.
Stem cells are undifferentiated or only partially differentiated
cells that have the capability to differentiate into a number of
progenitor and mature cell lineages and types. The term "stem
cells" can be used to refer to a cell type which is the progenitor
of a differentiation cellular lineage in a larger organism, such as
hematopoietic stem cell, or can refer to totally undifferentiated
stem cells which, at least in theory, have the ability to
differentiate into any of the tissues of the body. Stem cells are,
at a minimum, pluripotent, meaning that they have the potential to
differentiate into many different cell types, and may be
totipotent, meaning have the potential to differentiate into any
cell type of a mature organism of the species. Stem cell cultures
have been developed from a variety of tissue types and from a
number of different animals.
[0004] Recently, it has become possible to generate, culture and
maintain cultures of primate embryonic stem cells, including human
and rhesus embryonic stem cells. See, for example, U.S. Pat. No.
5,843,780 and U.S. Pat. No. 6,200,806 to Thomson. Primate embryonic
stem cells are stem cultures created from embryos that survive
indefinitely in culture and demonstrate the ability to
differentiate into the major tissue types of the primate body.
Primate embryonic stem cells can be maintained indefinitely in an
undifferentiated state in culture, or can be allowed to start a
differentiation process by which various of the cells become
committed to one or multiple developmental lineages. Typically, the
differentiation of stem cells into different tissue types begins
with the creation of embryoid bodies, which causes the stem cells
in the embryonic body to begin to differentiate into various cell
types.
[0005] A more differentiated type of human cell of scientific and
research interest is a human trophoblast. A trophoblast is a cell
which is a precursor of the cells which participate in the
formation of the human placenta. When an embryo begins
differentiation, at the stage of a blastocyst, the cells in the
inner cell mass are committed to form the cells which will become
the embryo, while the outer cells of the blastocyst become
committed to participate in the development of the placenta.
Trophoblast cells have been isolated before, but they are difficult
to isolate and have not been available for research in significant
amounts. Mouse trophoblast cell lines have been created from
blastocyst and post-implantation trophoblasts. Human trophoblast
cell lines have been created from transformed placental cells, but
techniques to create cultures of primate trophoblasts from
embryonic cells or stem cell lines have not yet been reported.
While human embryonic stem cells will spontaneously differentiate
into a number of differentiated cell types, including some
trophoblast cells, this phenomenon has not led to the creation of
useful cultures of trophoblast cells. In fact, mouse embryonic stem
cells appear to lack the ability to differentiate into trophoblast,
and hence, the supply of trophoblasts has always been extremely
limited. A replenishable supply of consistent trophoblast cells
would be very useful for many pharmaceutical investigations. In
particular, the exploration of contraceptive drugs targeting embryo
implantation and therapeutics preventing placenta-related birth
defects remain the topics of scientific investigation that can be
pursued with more ease provided that a source of primate
trophoblasts is available.
BRIEF SUMMARY OF THE INVENTION
[0006] The present invention is summarized in that a method to
induce primate stem cells to predominantly differentiate into human
trophoblasts includes the step of culturing the primate stem cells
in the presence of a protein trophoblast-inducing factor.
[0007] The present invention is also directed to uniform cultures
of primate trophoblast cells created by the method taught here.
[0008] The present invention is also directed to a method for
testing agents on placental cells in which the agents are exposed
to trophoblast cultures as described here.
[0009] It is an object of the present invention to enable the
creation of cultures of nearly pure trophoblasts in a uniform
consistent and reproducible manner.
[0010] It is a feature of the present invention in that it teaches
the first method known to cause primate stem cells in culture to
repeatedly, directly, individually and in synchrony predominantly
differentiate into a committed cell lineage.
[0011] Other objects, features and advantages of the present
invention will become apparent from the following
specification.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0012] FIG. 1 is a graphical illustration of the secretion of
hormones by trophoblast cells cultured according to the present
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0013] The present invention is premised on an observation. It has
been found by the inventors here that certain protein factors will
cause primate embryonic stem cells to differentiate directly into
trophoblast cells. The trophoblast cells are stable and exhibit all
of the cellular characteristics of placental precursor cells. Since
the creation and culture of primate and human embryonic stem cells
have become standardized and readily reproducible, this observation
makes possible for the first time the creation of a major class of
a single cell type (trophoblast cells) directly from a human or
other primate stem cell source, Without intervening creation of an
embryoid body. It has been found here that the protein factors that
cause direct differentiation of primate embryonic stem cells to
trophoblast cells include bone morphogenic protein 4 (BMP4) as well
as related protein factors such as BMP2, BMP7, and growth and
differentiation factor 5 (GDF5). Such a factor is here referred to
as a trophoblast-inducing factor.
[0014] The availability of human and other primate trophoblast
cells in reproducible quantities makes possible many investigative
studies on the behavior of placental cells. It is now possible to
have a reproducible and inexhaustible supply of placental precursor
cells. In particular, it is envisioned that the trophoblast cell
cultures can be used for chemical reaction studies to model the
behavior of placental cells for use in drug testing, both for
general toxicology as well as for specific effects on placental
cells. For example, agents, which would inhibit fertilized embryo
implantation in a uterus, i.e. birth control agents, can be
investigated by observing the effects of putative agents on the
trophoblast cell cultures.
[0015] This disclosure includes data which demonstrates that human
and other primate stem cells can be directly transformed into
trophoblast cells by treatment with a single protein factor. As
used herein, primate stem cells refers to human or other primate
undifferentiated cells which are at least pluripotent. The stem
cells used in the examples below originate from human and rhesus
embryos, and hence are known as primate embryonic stem cells.
Embryonic stem cells are stem cells derived from humans at some
stage of development. The method described here is, however,
equally applicable to human stem cells derived from other origins,
including embryonic germ line cells and stem cells isolated from
mature primate bodies. Note that the fact that human stem cells
will differentiate into trophoblast cells is unexpected based on
experience with mouse stem cells. Efforts to derive trophectoderm
tissue from mouse stem cells by manipulation of the external
culture environment have so far been unsuccessful, and when formed
into chimeras with intact pre-implantation embryos, mouse stem
cells rarely contribute to the trophoblast. The failure of mouse
embryonic stem cells to form trophoblast cells is consistent with
the theory that mouse embryonic stem cells are developmentally
similar to primitive ectoderm, which forms after delamination of
the primitive endoderm from the inner call mass and which no longer
contributes to the trophoblast lineage. The ability of human
embryonic stem cells to form trophoblast cells suggests a basic
difference between the development potential of mouse and human
embryonic stem cells.
[0016] Note that the method here involves the application of the
trophoblast-inducing factor directly to stem cells in culture,
without any intervening processes normally associated with
differentiation of stem cells. In particular, note that no stage of
transition to embryoid bodies is associated with this method. The
differentiation process for stem cells cultured by other means is
generally not uniform, in the sense that many different cell
lineages or cell types normally result. By contrast, the method
described here results in a mass differentiation of the stem cells
to a common differentiated cell type, trophoblast.
[0017] To demonstrate that the directed differentiation of stem
cells into trophoblast cells is due to the influence of the
trophoblast-inducing factor, it is possible to inhibit the action
of the trophoblast-inducing factor and observed the result. If BMP4
is the trophoblast-inducing factor, this protein can be inhibited
by soluble BMP4 receptor or by the antagonizing protein noggin.
That is, if one cultures primate stem cells with BMP4 only, the
stem cell culture will exhibit large scale directed differentiation
to trophoblast cell types. However, if one cultures a similar
primate stem cell culture with BMP4 and an inhibitor, such as the
soluble BMP4 receptor or noggin, the differentiation to trophoblast
cells will not occur.
[0018] Human embryonic stem cells in culture have a very
distinctive morphology. The cells are small, compact, and uniform,
have distinct cell membranes and cluster in groups. The
differentiation of stem cells into other cell types is a visible
process as the stem cells become larger and more diffuse.
Experienced technicians can recognize by cellular appearance of the
differentiated cells for many cell types. In the case of
trophoblast cells, the cells do become larger and flatten, and the
cells membranes becomes diffuse to invisible. However, to
supplement the status of the trophoblast cells, various
characterizing studies of the cells were undertaken. A gene
expression study using DNA microarrays was conducted to examine the
gene expression pattern of the cells. The secretion of placental
hormones by the cells was also examined. The results were
consistent with the identification of these differentiated cells as
trophoblast cells. This confirmed that the morphological
identification of these cells was correct.
EXAMPLES
[0019] A human embryonic stern cells line, H1, was cultured on a
Matrigel.TM.-coated plastic plate in medium that had been
conditioned on mouse embryonic fibroblasts (MEF) and supplemented
with basic fibroblast growth factor (bFGF) at 4 mg/ml. Human bone
morphogenic protein 4 (BMP4) (R&D Systems, Minneapolis, Minn.,
also the source for other recombinant proteins listed here) was
applied to the stem cells at concentrations of 1, 10 and 100 ng/ml
of culture medium. The stem cells were as a monolayer and not
aggregated in embryoid bodies. The H1 cells then underwent a dose
and time dependent morphological change, becoming spread out, flat,
thin and enlarged or elongated with their nuclei becoming smaller.
These changes are consistent with the morphology of trophoblast
cells. The morphological changes began with the cells at the edge
of each colony and spread inward from there. The changing
morphology became evident on day 2 for cultures treated with 100
ng/ml BMP4, day 3 or 4 for cultures treated with 10 ng/ml and days
4 to 5 for cultures treated with 1 ng/ml BMP4.
[0020] Similar experiments were conducted with other members of the
BMP4 protein signal family. Proteins which have been demonstrate to
activate a similar effect, to cause stem cells to change to
trophoblast cells, include BMP2, BMP7, and growth and
differentiation factor 5 (GDF5). Other proteins, including members
of the TGF superfamily, such as TGF beta 1 and activin, were found
not to activate this same morphological change in stem cells.
Similar morphological changes were observed on rhesus embryonic
stem cell lines treated with BMP4, BMP2, BMP7 and GDF5.
[0021] The change in morphology of the stem cells treated with the
trophoblast-inducing factor is consistent with the morphological
changes that occur in the development of an embryo where some cells
become committed to a lineage resulting in the placenta. In
addition to the morphological changes, the cells begin to express
transcription factors GATA2 and GATA3 and chorionic gonadatrophin
alpha and beta genes, all of which are expressed in trophoblasts
created by other means. The cells produce high amounts of placental
hormones including chorionic gonadatrophins, estradiol, and
progesterone. The cells continue to secrete these hormones
indefinitely. Flow cytometry of the cells demonstrate that the
cells are, at least predominantly, CG beta positive.
[0022] Experiments were also conducted in which antagonists of BMP
family factors were added to the culture at the same time as the
protein factor. It was found that if a soluble BMP receptor (at 100
ng/ml) or the BMP antagonizing protein noggin (at 300 ng/ml) were
added to the culture at the same time as for BMP4, the
morphological change in the stem cells was entirely prevented. This
demonstrates the specificity of the effect of the activating
protein factor.
[0023] Similar experiments were conducted on another stem cell
lines, named H9, with similar result in the production of
trophoblast cells. In addition, a similar experiment was conducted
on H1 cells cultured in the absence of bFGF, suggesting that the
effect is universal to human stem cells from various donors and is
not dependent on the presence of bFGF.
[0024] To further investigate the character of the trophoblasts,
cDNA microarrays were used to analyze genes differentially
expressed in the BMP4-treated cells and the untreated
undifferentiated H1 cells. Of the 43,000 cDNA genes examined on the
arrays, a cluster of only 19 clones, representing 14 genes, was
strongly upregulated during all the time points examined. Of these
14 genes, 11 have been previously characterized as related to the
development of trophoblast or placenta. Many of these genes encode
transcription factors, such as transcription factor AP-2 (TFAP2),
msh homeobox homolog 2 (MSX2), a suppressor of cytokine signaling 3
(SSI3), GATA binding proteins 2 and 3 (GATA2 and GATA3) and
hairy/enhancer-of-spli related with YPRW motif 1 (HEY1). By day 7
of treatment with BMP4, there was also observed a dramatic increase
in mRNA expression of many genes known to be expressed in
trophoblast or placenta, such as genes encoding CG-.alpha. and
CG-.beta. subunits, luteinizing homone-alpha and placental growth
factor. We also sued RT-PCR to observe enhanced expression of
trophoblast markers, including CG-.beta., glial cells missing-1
(GCM1), the non-classical HLA class 1 molecule HLA-G1, and CD9. All
of the top ten upregulated clones, representing 8 genes, in the
microarray analysis, with one exception, encode proteins or
peptides previously associated with genes expressed in trophoblast
cells. By contrast, after 7 days of BMP4 treatment, transcripts of
several genes highly expressed in pluripotent cells had declined,
such as those encoding the POU domain, class 5, transcription
factor 1 (POU5F1, also known as OCT4), and telomerase reverse
transcription factor (TERT).
[0025] To further confirm the character of the cells, the amount of
the placental hormones CG, estradiol and progesterone secreted into
the medium of the cells was examined. H1 cells treated with BMP4
showed markedly higher concentrations of each hormone as compared
to undifferentiated cells or cells differentiated in unconditioned
medium. FIG. 1 illustrates the time course in the increase of these
hormones in the cells exposed to BMP4 (CM+BMP4) as compared to
cells without BMP4 (CM) and cells permitted to differentiate in
unconditioned medium (UM).
* * * * *